The molecular nature of insulin resistance in human muscle is still incompletely defined. Recent results from mass spectrometry experiments performed to analyze serine/threonine phosphorylation of IRS-1 from insulin resistant human muscle now compel us to discover how changes in S/T phosphorylation in human insulin resistance affect the association of IRS-1 with its binding partners. Toward this, we propose 1) to use Surface Plasmon Resonance techniques to determine how site-specific serine/threonine phosphorylation of IRS-1 alters the kinetics of the interaction of IRS-1 with its binding partners. We will employ surface Plasmon resonance techniques to quantify the kinetics of association of IRS-1 binding partners with synthetic tyrosine- phosphorylated IRS-1 peptides that also are phosphorylated at candidate S/T residues informed by our previous studies. In addition, reversible acetylation of proteins is gaining prominence as a mechanism that regulates mitochondrial. Preliminary data indicate that acetylation of mitochondrial proteins in humans is regulated by muscle contraction and is dysregulated in insulin resistance. Therefore we also propose 2) to use a combination of clinical research and mass spectrometry techniques to determine how the cytosolic and mitochondrial protein acetylomes are regulated by muscle contraction in insulin sensitive and resistant human volunteers. We will test the hypothesis that mitochondrial protein acetylation is decreased to a greater degree following a bout of exercise in insulin sensitive than in insulin resistant human muscle. Using these techniques we also propose 3) to determine how acetylation of mitochondrial adenine nucleotide translocase (ANT1) at lysines 10, 23, and 92 regulates ANT1 structure and function. Finally, we propose 4) to use a combination of molecular modeling and in vitro assays together with the approach developed in Aim 3 to characterize the role of acetylation in other mitochondrial proteins. Protein targets for this aim will be prioritized based on the potential role of the protein in insulin resistance or mitochondrial function as well as dysregulation of its acetylation state in insulin resistant muscle. PUBLIC HEALTH RELEVANCE: Insulin resistance underlies the major public health problems of obesity, type 2 diabetes mellitus, and cardiovascular disease. Understanding the molecular nature of this abnormality in humans will be a key to developing and assessing the effectiveness of new treatments for these diseases.